Laser scanning microscope 700

HEK293 cells were grown on coverslips in 24-well plates coated with 50 μg/mL OptiCol™ human collagen type I (Cell Guidance Systems) to a confluency of 50–70%. Cells were transfected with pVLDLR_mGFP, pApoER2_mGFP, pmCherry-N1_ApoER2 + pVLDLR_mGFP or mGFP. After 24 h, 25 μl of concentrated RCM (cRCM) was added to the wells. Cells were incubated with cRCM at 37°C for 5 min, washed twice with cold PBS and fixed with 4% formaldehyde for 15 min at room temperature. Fixed cells were washed three times with cold PBS, incubated in blocking solution (1% BSA in PBS) for 30 min at room temperature and overnight with primary antibody (anti-Reelin, G10) at 4°C. On the next day, samples were washed three times with cold PBS and incubated with secondary antibody, goat anti-mouse IgG DyLight633 (ThermoScientific) for 1 h at RT. Afterwards, cells were washed three times with cold PBS and incubated 5 min in DAPI solution (5 μg/ml), washed again, incubated in quenching buffer (100 mM glycine) for 15 min. After the final wash with H₂0, coverslips were mounted using ibidi Mouting Medium (ibidi) and sealed with nail polish. Slides were analyzed using a confocal fluorescence microscope (laser-scanning microscope 700, Zeiss) and the corresponding ZEN software.

Cells were cultured on Lab-Tek II Chamber Slides (Nalgene Nunc International, Rochester, 154534). The Premo^TM Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Life Technologies, P36239) was used to monitor autophagy flux. The cells were treated, followed by fixation in a 4% formaldehyde solution. Fluorescent images were obtained using the Laser Scanning Microscope 700 (Carl Zeiss).

The mFER coding sequence was cloned in-frame and upstream of the sequence encoding the N terminus of YFP, while the SHOU4, FBH3, ABI5, and DDL coding sequences were cloned in-frame and upstream of the sequence encoding the C terminus of YFP. All resulting plasmids were individually introduced into Agrobacterium (Agrobacterium tumefaciens) (strain GV3101). Positive Agrobacterium colonies were cultured in liquid LB medium containing 0.2-mM acetosyringone for 1–2 days. Cells were collected by centrifugation, washed, and resuspended in infiltration buffer (10-mM MgCl₂, 10-mM MES, pH 5.7, 0.2-mM acetosyringone) to a final optical density (OD) measured at a wavelength of 600 nm (OD₆₀₀) of 0.9. Agrobacteria carrying appropriate pairs of nYFP and cYFP constructs were mixed in a 1:1 ratio and infiltrated into the lower surface of N. benthamiana leaves from 2-month-old plants grown on soil. After 36 h, YFP signals were detected using a Zeiss Laser Scanning Microscope 700 (LSM700).

Human eSCs were incubated in DMEM containing 1.0 g/L glucose supplemented with 10% FBS. The medium was then replaced with DMEM (0.1% FBS), after which the cells were incubated at 37 °C in an atmosphere of 5% CO₂ for 18 h to minimize cell proliferation. An artificial wound was created by disrupting the monolayer using a sterile plastic pipette tip (200 µL). A migration assay was then performed in the presence or absence of SM at 6, 12, and 24 h. The cells were then stained using the CytoPainter Cell Tracking Staining Kit (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol. Images were captured using a laser Scanning Microscope 700 (Carl Zeiss, Oberkochen, Germany) equipped with a 5× objective. Cell migration was measured as the percentage of the remaining wound area relative to the cell-free area of the initial scratch. The number of migrated cells was calculated using an ImageJ cell counter. All experiments were performed in at least triplicate.